The present invention relates to methods and compositions for detecting mutations associated with thrombopathy. In particular, the invention relates to methods and composition for detecting mutations in the CalDAG-GEF1 gene that are associated with thrombopathy.
The platelet integrin alphaIIb-beta3, also known as the platelet glycoprotein complex IIb-IIIa, mediates platelet aggregation by binding the dimeric ligand fibrinogen. In unactivated platelets, the integrin is in a low-affinity/avidity state for the binding of fibrinogen. The conversion from a low-affinity to a high-affinity state is mediated by signal transduction proteins mobilized in response to agonists binding to specific receptors on the platelet surface; this sequence of events is termed “inside-out” signaling (R. O. Hynes, Cell 110:673-687 (2002); S. J. Shattil and P. J. Newman, Blood 104:1606-1615 (2004)). Examples of agonists that can induce “inside-out” signaling and thus change the affinity of the integrin for fibrinogen include ADP, collagen, thromboxane, and thrombin. Once fibrinogen has bound to alphaIIb-beta3, signal transduction events referred to as “outside-in” signaling occur, which increase the avidity of the integrin for fibrinogen and are accompanied by integrin receptor clustering.
Rap1 is a Ras-related low-molecular-weight guanine-nucleotide-binding protein (GTPase) that is ubiquitously expressed in high levels in platelets, neutrophils, and brain (J. L. Bos, EMBO J 17:6776-6782 (1998)). Rap1 seems to play an important role in several cell processes, including cell proliferation and differentiation, platelet, neutrophil, and B-cell activation, induction of T-cell anergy, and the regulation of the respiratory burst in neutrophils (J. L. Bos, All in the family? New insights and questions regarding interconnectivity of Ras, Rap1 and Ral, EMBO J 17:6776-6782 (1998)). Rap1 is activated by many different stimuli in different cell types and is thus a shared (common) component in signaling. Receptor-signaled generation of second messengers, including calcium, cAMP, and DAG can lead to the direct activation of Rap1-specific guanine nucleotide exchange factors (GEFs).
Platelets contain high levels of Rap1b. In platelets, Rap1 is involved in integrin activation and is activated by the binding of GTP and inactivated by the hydrolysis of bound GTP to GDP. In quiescent platelets, Rap1b is primarily localized to the plasma membrane (E. G. Lapetina et al, Proc Natl Acad Sci USA 86:3131-3134 (1989)). After activation, Rap1b relocalizes to the actin cytoskeleton (T. H. Fischer et al., J Biol Chem 265:19405-19408 (1990)). Platelet agonists that stimulate fibrinogen binding to platelet integrin alphaIIb-beta3 also stimulate binding of GTP to Rap1b resulting in its rapid activation (B. Franke et al., EMBO J 16:252-259 (1997); M. Torti and E. G. Lapetina, Proc Natl Acad Sci USA 89:7796-7800 (1992),). Most physiologic agonists (including ADP and collagen) that stimulate Rap1b activation do so through stimulation of Gi-coupled receptors (P. Lova et al., J Biol Chem 277:12009-12015 (2002); D. Woulfe et al., J Biol Chem 277:23382-23390 (2002)). Platelet adhesion mediated by binding of the platelet integrin alpha2-beta1 to collagen initiates outside-in signaling pathways that also result in activation of Rap1b (B. Bernardi et al, Blood 107:2728-2735 (2006)). Agonists that activate platelets through Gq-coupled receptors, including thromboxane, or through cross-linking of FcγRIIA, which is a tyrosine kinase-based pathway, rely on binding of secreted ADP, which in turn binds to the Gi-coupled ADP receptor P2Y12 (P. Lova et al., J Biol Chem 277: 12009-12015 (2002)). The importance of Rap1b for normal platelet function and hemostasis was demonstrated recently in a knockout mouse model. Rap1b null mice exhibited markedly impaired platelet function and experienced 85% embryonic and perinatal lethality primarily caused by hemorrhage-related events (M. Chrzanowska-Wodnicka et al, J Clin Invest 115:680-687 (2005)).
Calcium-Diacylglycerol Guanine Nucleotide Exchange Factor I (CalDAG-GEFI) is a GEF that activates Rap1b. CalDAG-GEFI contains 4 major domain structures: (1) a Ras exchanger motif (REM) domain common to Ras family GEFs; (2) a cdc25-like GEF domain (catalytic domain); (3) two EF hand domains for interaction with calcium; and (4) a C1 domain for interaction with DAG and phorbol esters. Guanine nucleotide exchange factors promote nucleotide release from GTPases. GDP is not preferentially released over GTP; cellular concentrations of GTP are generally 10 times higher than concentrations of GDP; thus, GTP has a higher likelihood of rebinding to the GTPase than GDP (P. A. Boriack-Sjodin et al., Nature 394: 337-343 (1998)). The exchange of GTP for GDP promotes the activity of GTPases. CalDAG-GEFI knockout mice exhibit severely impaired platelet aggregation and release responses to most agonists, including ADP, collagen, thromboxane, and the calcium ionophore A23187 (J. R. Crittenden et al., Nat Med 10:982-986 (2004)). CalDAG-GEFI effects are likely caused by affinity/avidity modulation of integrin alphaIIb-beta3 via activation of Rap1b (A. Bertoni et al, J Biol Chem 277 (2002), pp. 25715-25721). It is possible, however, not all the CalDAG-GEFI effects on platelet function reported in the knockout mice may be linked to activation of Rap1b.
Intrinsic platelet function disorders have been described in Basset hounds and Eskimo Spitz dogs (I. B. Johnstone and F. Lotz, Can Vet J 20:211-215 (1979); J. L. Catalfamo et al., Blood 67:1568-1577 (1986); M. K. Boudreaux et al., J Vet Int Med 8:93-98 (1994)). A platelet defect has been recognized in Landseers, a European dog breed related to the Newfoundland breed; however, biochemical and functional descriptions of the disorder are lacking. Affected Basset hounds, Spitz dogs, and Landseers experience epistaxis, gingival bleeding, and petechiation on mucous membranes and skin. Platelet number, plasma von Willebrand factor concentration, and function and coagulation screening assays are normal, whereas bleeding time tests are prolonged. In contrast to Rap1b null mice, reduced litter sizes or high neonatal lethality have not been described with these disorders in dogs.
The platelet disorders in Basset hounds and Spitz dogs have been well characterized, and they are essentially similar at the functional level. Platelet aggregation responses to ADP, collagen, calcium ionophore A23187, and platelet activating factor (PAF) are severely impaired. In response to thrombin, their platelets exhibit a characteristic lag phase with normal maximal extent aggregation. Epinephrine enhances the sensitivity of thrombopathic platelets to ADP, but the aggregation response is still reduced compared with normal platelets. Thrombopathic Basset hound intraplatelet fibrinogen content and concentrations of membrane glycoproteins IIb and IIIa are normal. Platelets from affected Basset hounds and Spitz dogs support normal clot retraction. cDNA sequences and coding areas of genomic DNA for platelet glycoproteins IIb and IIIa obtained from affected Basset hounds and heterozygous Landseers are identical to sequences obtained for normal dogs except for the presence of benign polymorphisms (Boudreaux, unpublished findings). The similarity of the platelet function disorder described in affected dogs to that identified in CalDAG-GEFI knockout mice, combined with the lack of embryonic or neonatal lethality described in the CalDAG-GEFI mouse knockout, prompted the evaluation of the gene encoding CalDAG-GEFI in affected Basset hounds, in an affected Eskimo Spitz dog, and in an affected Landseer. Three distinct CalDAG-GEFI gene mutations were identified in each breed evaluated.
Intrinsic platelet disorders also have been described in Simmental cattle (Steficek et al., J Vet Diagn Invest 5:202-7 (1993); Searcy et al., Can J. Vet. Res. 54:394-396 (1990). A distinct CalDAG-GEF1 gene mutation likewise was identified in Simmental cattle.
All mutations were located in portions of the gene encoding the highly conserved catalytic unit. The changes are considered significant and would result in either lack of synthesis, enhanced degradation, or marked impairment of protein function.